Cellular architecture of Treponema pallidum: novel flagellum, periplasmic cone, and cell envelope as revealed by cryo electron tomography.

Ethics Statement

All procedures involving rabbits were reviewed and approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston.

Cryo-ET of intact T. pallidum T. pallidum subsp. pallidum

Nichols was extracted from rabbit testes after intratesticular infection; in some experiments, the organisms were further purified by Percoll density gradient centrifugation as described previously 20 . Freshly prepared, motile T. pallidum was centrifuged and resuspended in 20 μl phosphate-buffered saline (PBS) at a final concentration ~2×10 9 cells/ml. After mixing with 15 nm gold clusters, 4 μl T. pallidum samples were deposited onto freshly glow-discharged holey carbon grids for 1 min. The grids were blotted with filter paper and rapidly frozen in liquid ethane maintained at −180°C using a gravity-driven plunger apparatus as previously described 44 . The resulting frozen-hydrated specimens were imaged at −170 °C using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a 4K × 4K CCD (16 megapixel) camera (TVIPS; GMBH, Germany). The microscope was operated at 300 kV with a magnification of 31,000×, resulting in an effective pixel size of 2.8 Å. Using the FEI “batch tomography” program, low dose single-axis tilt series were collected from each bacterium at −4 to −6 μm defocus with a cumulative dose of ~100 e − /Å 2 distributed over 65 images, covering an angular range from −64° to +64°, with an angular increment of 2°. A 30 μm objective aperture and 2×2 binning of pixels was used to enhance the image contrast at this defocus level. Tilted images were initially aligned with respect to each other using fiducial markers and the IMOD software package 68 . After further refinement using projection-matching, 3-D tomograms were reconstructed using weighted back-projection implemented in the package Protomo 69 . In total, 304 cryo tomograms from T. pallidum cells were reconstructed. The effective pixel size of the tomographic reconstructions is 5.6 Å, and the effective in-plane resolution is better than 4 nm, based on the direct separation of lipid bilayers. 2×2×2 binning of these tomograms was used for the visualization of 3-D images and the preparation of figures. Outer membrane removal, surface proteolysis and lysozyme treatment Percoll-purified T. pallidum organisms in PBS (~1 × 10 9 ) were centrifuged at 21000 × g for 1 m, resuspended in 500 μl PBS with 5 mM MgCl 2 , and pipetted vigorously ~20 times. This process was repeated, As a result of this treatment, the outer membrane was removed from the majority of T. pallidum organisms. Surface proteolysis was carried out on this preparation by treatment with Proteinase K (0.4 mg/ml) for 40 m at ambient temperature as described in 44 . The same preparation was treated in parallel with lysozyme (0.6 mg/ml).

Immunogold labeling of flagellar filaments Percoll-purified

T. pallidum cells partially disrupted by repeated centrifugation as described above were applied to carbon coated grids and incubated with PBS containing 5% bovine serum albumin (BSA) for 30 min, followed by washing with PBS-5% BSA. Grids were placed on a 30 ul drop of primary rabbit antisera against either FlaA or FlaB1 32 diluted 1:20 in PBS-5% BSA for 60 m, followed by washing again with PBS-5% BSA three times. The samples were stained with gold goat anti-rabbit immunoglobulin G (IgG) (Jackson ImmunoResearch) diluted 1:20 in PBS-5% BSA for 60 m followed by washing in PBS with 2% BSA. The grids were washed with water before staining with 1% uranyl acetate. Samples were viewed in a JEM1200 electron microscope. Subvolume averaging of flagellar motor and filament The subvolume processing of flagellar motors was carried out as described in 44 . Briefly, the positions and orientations of each flagellar motor in each tomogram were determined manually. Subvolumes (256×256×256 voxels) containing entire flagellar motors were extracted from the original tomograms. In total, 830 flagellar motor volumes were extracted from 273 tomograms and were further used for 3-D alignment, classification and averaging. The resolution of the flagellar motor structure is 4.0 nm based on the Fourier shell correlation (cutoff 0.5). A total of 4,166 segments (192×192×96 voxels) of flagellar filaments were manually identified and extracted from 55 reconstructions. The initial orientation was determined by using two adjacent points along the filament. The first one is proximal to the flagellar hook, and the polarity of the filament was preserved during the alignment process. After obtaining the asymmetric reconstruction of the flagellar filament, the helical symmetry of the short segment was determined and imposed. The helical model was then used as the reference for further 3-D alignment and averaging.

3-D Visualization

Tomographic reconstructions were visualized using IMOD 68 and surface rendering of flagellar structures was carried out with the software package UCSF Chimera 70 . Reconstructions of several T. pallidum organisms were segmented manually using 3-D modeling software Amira (Visage Imaging). 3-D segmentation of the flagellar filaments, cytoplasmic filaments, outer and cytoplasmic membranes, and unique cone-shaped structure were manually constructed. The surface model from the averaged flagellar motor was computationally mapped back into the original cellular context as described 71 .

Scanning Probe Microscopy

SPM was carried out using a Digital Instruments/Veeco MultiMode ™ instrument equipped with a Nanoscope IIIa SPM controller. A cantilever probe with a spring constant of 40 N/m and an inherent resonance (f 0 ) of ~300 kHz was used. Freshly extracted T. pallidum (6 × 10 6 /ml) in PBS was centrifuged at 500×g for 5 min to remove host cell debris and fixed in 3% glutaraldehyde. A 10 μl sample was applied to a mica support and air dried. The specimen was rinsed 3 times with distilled water and air dried prior to SPM. Scanning was performed in the tapping mode with a scan rate of 1 line/sec.

Supplementary Material 01 Figure S1 The T. pallidum periplasmic flagella and flagellar motor in situ . The flagella are located in the periplasmic space, while the motors are embedded in the cytoplasmic membrane. (A) and (C) are two organisms with the two most frequently observed morphologies - curved and straight. (B) and (D) are enlarged views of (A) and (C), respectively. White arrows indicate the location of a flagellar motor. Black arrows indicate the location of periplasmic flagella. Only one or two motors are visible in each tomogram section. The scale bar is 100nm. 02 Figure S2 Thin flagellar filaments lack the FlaA surface layer. Flagellar filaments were released from cells by repeated centrifugation and resuspension prior to immunogold labeling. (A and C) Immunolabeling with rabbit anti-FlaA antiserum. 12nm gold particles were primarily attached to 20nm thick flagellar filaments after FlaA antibody labeling. Few gold particles were associated with 14 nm thin flagellar filaments. (B) Labeling with rabbit anti-FlaB1 antiserum. Gold particles were mainly attached to thin filaments after anti-FlaB1 antibody treatment. (D) Control without primary antibody. 03